Luminescence quenching is a well-established optical procedure for the determination of the concentrations of analytes which are themselves quenchers of excited states. The simplest and least expensive procedure for such determinations involves the use of fluorescence intensity measurements. However, such procedures require frequent recalibration due to photobleaching of the fluorophore, and may be inaccurate or imprecise due to light scattering within the medium and by the filtering effects of substances in the sample which absorb light at the excitation or emission wavelengths. Examples of fluorescence intensity procedures which suffer from some or all of the above deficiencies are U.S. Pat. No. 4,476,870 (Peterson et. al. ) and U.S. Pat. No. 5,186,173 (Zuckerman) for the determination of dioxygen concentration levels. The determination of luminescence lifetime (decay) rather than intensity overcomes the deficiencies of intensity-based procedures. Luminescence lifetime is typically measured by one of two time-resolved procedures, viz., pulse fluorometry or phase modulation fluorometry. Examples of such approaches are U.S. Pat. No. 5,039,219 (James et. al.) in the case of pulse fluorometric procedures and U.S. Pat. No. 5,317,162 (Pinsky et. al.) as well as U.S. Pat. No. 5,281,825 (Berndt et. al.) in the case of phase modulation fluorometry. However, when applied to fluorophores with lifetimes in the nanosecond range the costs for implementing such techniques can be prohibitive, and have prevented their use in clinical laboratory instruments. That is, the cost of pulse lasers and gating devices such as microchannel plate intensifiers and photon counters for pulse fluorometry, and devices to modulate excitation light in the 10-100 MHz. range for phase systems have restricted such procedures to the research laboratory. Similarly, such direct lifetime systems suffer from a further deficiency in addition to high cost, i.e., they become increasingly less accurate as luminescence lifetime shortens with increasing concentrations of the quenching analyte.
In pending U.S. Pat. Ser. No. 08/231,191 (Zuckerman) a novel procedure for the indirect, steady-state determination of luminescence lifetime was disclosed for the case of the analyte dioxygen. The methodology, based upon the determination of luminescence anisotropy, overcomes the deficiencies in time-resolved determinations of luminescence lifetimes. In addition, to one skilled in the art it would be obvious that the method disclosed in the above noted pending patent application is a general methodology which may be applied to the determination of the concentration levels of numerous analytes of biologic and physical import. Herein, the general methodology is further detailed for the case of analytes which quench excited states, and a novel adaptation of this procedure is disclosed which allows the method to be extended to the measurement of concentrations of analytes which do not themselves quench excited states.